Production, Purification, and Characterization of ¹⁵N-Labeled DNA Repair Proteins as Internal Standards for Mass Spectrometric Measurements.

Abstract

Oxidatively induced DNA damage is caused in living organisms by a variety of damaging agents, resulting in the formation of a multiplicity of lesions, which are mutagenic and cytotoxic. Unless repaired by DNA repair mechanisms before DNA replication, DNA lesions can lead to genomic instability, which is one of the hallmarks of cancer. Oxidatively induced DNA damage is mainly repaired by base excision repair pathway with the involvement of a plethora of proteins. Cancer tissues develop greater DNA repair capacity than normal tissues by overexpressing DNA repair proteins. Increased DNA repair in tumors that removes DNA lesions generated by therapeutic agents before they became toxic is a major mechanism in the development of therapy resistance. Evidence suggests that DNA repair capacity may be a predictive biomarker of patient response. Thus, knowledge of DNA-protein expressions in disease-free and cancerous tissues may help predict and guide development of treatments and yield the best therapeutic response. Our laboratory has developed methodologies that use mass spectrometry with isotope dilution for the measurement of expression of DNA repair proteins in human tissues and cultured cells. For this purpose, full-length (15)N-labeled analogs of a number of human DNA repair proteins have been produced and purified to be used as internal standards for positive identification and accurate quantification. This chapter describes in detail the protocols of this work. The use of (15)N-labeled proteins as internal standards for the measurement of several DNA repair proteins in vivo is also presented.